microrna-9a ensures the precise specification of sensory...

MicroRNA-9a ensures the precisespecification of sensory organ precursorsin DrosophilaYan Li,1 Fay Wang,1 Jin-A Lee, and Fen-Biao Gao2

Gladstone Institute of Neurological Disease and Department of Neurology, University of California at San Francisco,San Francisco, California 94158, USA

MicroRNAs (miRNAs) have been implicated in regulating various aspects of animal development, but theirfunctions in neurogenesis are largely unknown. Here we report that loss of miR-9a function in the Drosophilaperipheral nervous system leads to ectopic production of sensory organ precursors (SOPs), whereasoverexpression of miR-9a results in a severe loss of SOPs. We further demonstrate a strong genetic interactionbetween miR-9a and senseless (sens) in controlling the formation of SOPs in the adult wing imaginal disc.Moreover, miR-9a suppresses Sens expression through its 3� untranslated region. miR-9a is expressed inepithelial cells, including those adjacent to SOPs within proneural clusters, suggesting that miR-9a normallyinhibits neuronal fate in non-SOP cells by down-regulating Sens expression. These results indicate thatmiR-9a ensures the generation of the precise number of neuronal precursor cells during development.

[Keywords: MicroRNA; SOP; Senseless; Drosophila; PNS]

Supplemental material is available at http://www.genesdev.org.

Received July 6, 2006; revised version accepted August 22, 2006.

The peripheral nervous system (PNS) is essential for ani-mals to detect and relay environmental stimuli to cen-tral neurons for information processing. It is largely un-known what ensures the development of precise num-bers of sensory organs for a particular external sensorycue. The Drosophila PNS has been used as an excellentmodel system for dissecting the genetic programs thatcontrol sensory organ formation (Ghysen and Dambly-Chaudiere 1993; Jan and Jan 1993; Modolell 1997). Inembryonic abdominal segments, external sensory (ES)organs and chordotonal (CH) organs contain single-den-drite neurons associated with support cells and functionin receiving mechanical stimuli (Campos-Ortega andHartenstein 1985; Ghysen et al. 1986). In contrast, mul-tidendritic (MD) neurons elaborate highly branched den-drites underneath the epidermis (Bodmer and Jan 1987;Gao et al. 1999) that probably function as stretch, touch,or other sensory receptors (Ainsley et al. 2003; Liu et al.2003; Tracey et al. 2003). In adult flies, most externalsensory organs have one neuron, one hair cell, and a fewsupport cells (Campos-Ortega and Hartenstein 1985).

Precise numbers of sensory organs are generatedthrough similar developmental processes in embryos andadults (Campos-Ortega and Hartenstein 1985). For ex-

ample, in embryonic/larval abdominal segments, eachdorsal cluster contains four ES organs and eight MD neu-rons. Some ES neurons are generated from sensory organprecursors (SOPs) that also produce MD neurons (ES–MD lineage), while other lineages produce ES but notMD neurons (Brewster and Bodmer 1995; Vervoort et al.1997). Adult flies have four macrochaetes on the notumand a well-defined number of sensory bristles on thewing margin. All cells in each adult sensillium are gen-erated from a single SOP in two or three rounds of asym-metric cell division (Hartenstein and Posakony 1989;Bardin et al. 2004).

The selection of SOPs from early ectoderm beginswith the proneural cluster, which consists of a smallnumber of cells that express proneural genes encodingthe basic helix–loop–helix (bHLH) proteins (Achaete,Scute, Asense, Atonal, and Amos), rendering those cellscompetent to develop into SOPs (Romani et al. 1989;Cubas et al. 1991; Skeath and Carroll 1991; Jarman et al.1993; Ruiz-Gomez and Ghysen 1993; Goulding et al.2000; Huang et al. 2000). It is thought that the cells withthe highest level of proneural proteins are selected asSOPs, which express a higher level of Delta and activateNotch in neighboring cells (Goriely et al. 1991; Artava-nis-Tsakonas et al. 1999). Activation of Notch initiates asignaling cascade in which Suppressor of hairless [Su(H)]and Enhancer of split [E(spl)] complexes are involved tosuppress neuronal fate in non-SOP cells (Knust et al.1992; Schweisguth and Posakony 1992).

1These authors contributed equally to this work.2Corresponding author.E-MAIL [email protected]; FAX (415) 355-0824.Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.1466306.

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One of the genes activated by bHLH proteins, sense-less (sens), encodes a transcription factor with four zincfingers whose expression is dynamically regulatedwithin the proneural clusters (Nolo et al. 2000). Withinthe proneural cluster, a high level of Sens is required toup-regulate and maintain the proneural gene expressionin SOPs (Nolo et al. 2000), while a low level of Sensrepresses the transcription of proneural genes in adjacentcells (Jafar-Nejad et al. 2003). This dual function of Sensas an activator or suppressor suggests that the properlevel of Sens is required to control the formation of theprecise number of SOPs. However, how differential ex-pression levels of Sens in SOPs and adjacent cells areensured remains unknown.

In recent years, post-transcriptional regulation bymicroRNAs (miRNAs) has emerged as an importantmechanism for controlling gene expression in animal de-velopment (Carrington and Ambros 2003; Bartel 2004;He and Hannon 2004; Alvarez-Garcia and Miska 2005;Carthew 2006). miRNAs are endogenous noncodingsmall RNAs 21–23 nucleotides (nt) in length. SeveralmiRNAs have been implicated in nervous system devel-opment, with roles ranging from regulating left/rightneuronal asymmetry in Caenorhabditis elegans, photo-receptor formation in Drosophila, and brain morphogen-esis in zebrafish, to neuronal differentiation in mammals(Johnston and Hobert 2003; Chang et al. 2004; Giraldezet al. 2005; Li and Carthew 2005; Vo et al. 2005; Krichev-sky et al. 2006; Schratt et al. 2006). However, the preciseroles of miRNAs in early neurogenesis have not beenstudied yet. Moreover, only a few miRNAs have beenstudied using loss-of-function approaches in Drosophila(e.g., Brennecke et al. 2003; Xu et al. 2003; Kwon et al.2005; Li and Carthew 2005; Sokol and Ambros 2005;Teleman et al. 2006).

Here, through both loss-of-function and gain-of-func-tion in vivo analyses, we found that miR-9a is requiredto ensure the generation of precise numbers of sensoryorgans in Drosophila embryos and adults. To accomplishthis regulatory function, miR-9a down-regulates the ex-pression of Sens through its 3� untranslated region (UTR)to ensure the differential expression of Sens in SOPs andadjacent epithelial cells. These findings provide new evi-dence that miRNAs function at the translational level toensure the appropriate level of gene expression duringdifferent developmental processes.

Results

Generation of miR-9a mutant flies

To understand the roles of miRNAs in neural develop-ment, we chose Drosophila miR-9a for further analysisfor the following reasons. First, the Drosophila homologof the human fragile X mental retardation protein affectsthe formation of terminal dendritic branches (Lee et al.2003) and is associated with miRNAs and the RNA-in-duced silencing complex, implicating miRNAs in hu-man mental disorders (Caudy et al. 2002; Ishizuka et al.2002). Second, miR-9a is one of the miRNAs that are

specifically and highly expressed in the mammalianbrain, suggesting a unique role in neural developmentand/or function (Lagos-Quintana et al. 2002). Third, ma-ture miR-9a is 100% conserved at the nucleotide levelfrom flies to humans (Fig. 1A; Aravin et al. 2003), indi-cating an evolutionarily conserved function. Moreover,in contrast to the mouse genome, which has three miR-9a precursors located on different chromosomes (Lagos-Quintana et al. 2002), the Drosophila genome has onlyone (Aravin et al. 2003; Sempere et al. 2004), providing agood opportunity to study the function of this miRNA.

The Drosophila miR-9a precursor is located on the leftarm of the third chromosome 76B7, 10 kb to the right ofgene CG9300 and 6.4 kb to the left of gene CG9262 (Fig.1B). To generate loss-of-function mutants of miR-9a, weused ends-out homologous recombination (Gong andGolic 2003) to replace the 78-nt long miR-9a precursorDNA sequence with the white gene (Fig. 1C). To screenfor potential miR-9a mutants, we used PCR analysiswith two primers flanking miR-9a. These primers pro-duced a 0.5-kb fragment when wild-type genomic DNAwas used as the template and a 4-kb fragment if miR-9awas replaced by white (Fig. 1D). From two independentgene-targeting experiments, we generated five mutantlines in which homologous recombination occurred atthe miR-9a locus (Fig. 1D). To confirm that the expres-sion of miR-9a was indeed abolished in mutants, we per-formed Northern blot analysis that showed the absenceof miR-9a in all five mutant lines (Fig. 1E). Lines J22 andE39, generated from two independent targeting events,were selected for further analysis.

Loss of miR-9a function results in ectopic sensoryneurons in Drosophila embryos and larvae

Both miR-9aJ22 and miR-9aE39 mutant flies developed toadulthood at the expected Mendelian ratio and were fer-tile, suggesting that miR-9a activity is not essential forsurvival or fertility. However, when we examined themorphology of PNS neurons, we found unexpectedlythat some of the MD neurons were duplicated. Nor-mally, Drosophila embryos and larvae have 12 sensoryneurons in each dorsal cluster in the abdominal seg-ments (Fig. 2A), including eight MD neurons. At the lar-val stage, each MD neuron has a unique dendriticbranching pattern that is fairly consistent from segmentto segment and among different animals (Bodmer and Jan1987; Grueber et al. 2002; Sweeney et al. 2002). Using aspecific driver, Gal4221, we examined two MD neurons,ddaE and ddaF (Sweeney et al. 2002), which extendsmooth dendrites toward the posterior and anterior, re-spectively (Fig. 2B). Twenty-nine percent of miR-9aJ22

(n = 62) and 37% of miR-9aE39 mutant third instar larvae(n = 27) contained an extra ddaE or ddaF neuron in one ortwo but not other hemisegments (Fig. 2C,D), while noneof the wild-type larvae had this phenotype (n = 24). Asimilar phenotype was also seen in vpda, a ventral MDneuron that also extends simple and smooth dendriticbranches (Supplementary Fig. S1).

The frequent observation of two-cell clones generated

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by mosaic analysis with a repressible cell marker(MARCM) (Lee and Luo 1999) that contained an ES neu-ron and either a ddaE neuron or a ddaF neuron (Sweeneyet al. 2002) suggests that ddaE and ddaF neurons are gen-erated from the ES–MD lineages. To examine whetherthe duplicated neurons in miR-9a mutants resulted fromcell fate transformation within the ES–MD lineage orfrom an ectopic SOP, we counted neurons and supportcells of A5–A6 dorsal clusters in embryos. As shown byimmunostaining, each wild-type dorsal cluster con-tained 12 Elav+ neurons (eight MD and four ES neurons)and eight Cut+/ELAV− cells (external cells, Fig. 2E). Inthe miR-9a mutant embryos, more cells were observedin some dorsal clusters. Among the four extra cells, therewere two Elav+ neurons accompanied by two Cut+/ELAV− external cells (Fig. 2F), suggesting that an extraSOP was generated. These findings suggest that miR-9aaffects the precise production of SOPs during Drosophilaembryonic development.

To understand how miR-9a is involved in SOP speci-fication, we performed in situ hybridization with a probefor the miR-9a precursor. The earliest expression of miR-9a was at stage 5 and was observed in all cells exceptmost ventral cellular blastula (data not shown). Aftergastrulation at stage 7, miR-9a expression persisted in allepithelial cells except the ventral ectoderm (Fig. 2G). Ec-todermal epithelial cells continued to express miR-9a,and at stage 10–13, strong expression of miR-9a was seenat the ectoderm of the segmental boundaries and stomo-deum (Fig. 2H). During this stage, embryonic PNS SOPsare derived from the epidermal cells. Consistently, weobserved a similar embryonic expression of miR-9a with

a probe made out of the mature miR-9a (data not shown),which was also reported independently during the courseof our work by Stark et al. (2005).

miR-9a controls the generation of sensory organsin adult flies

The increased production of sensory neurons in miR-9amutant embryos/larvae prompted us to further examinethe sensory organ formation in adult flies. Wild-type an-terior wing margins have a fixed number of regularlyspaced sensory bristles (Fig. 3A�). About 40% of miR-9amutants flies showed ectopic sensory bristles on anteriorwing margins (Fig. 3B�), while flies overexpressing miR-9a had a marked decrease in the number of bristles (datanot shown). miR-9a mutant flies also exhibited a poste-rior wing margin defect, which was 100% penetrant (Fig.3B). To confirm that the wing phenotype was caused bythe loss of miR-9a activity, we performed a genetic res-cue experiment. Expressing the miR-9a precursor by vg-Gal4, which drives gene expression in the thin strip ofdorsal–ventral boundary of wing imaginal discs, in themiR-9a mutant background completely rescued the de-fects on posterior wing margins (n = 152, Fig. 3C). Thebristle defect on the anterior margin of miR-9a mutantswas also largely rescued (Fig. 3C�).

Besides the wing phenotype, miR-9a mutant flies alsodisplayed an ectopic production of sensory organs on thenotum. Each SOP generates five different cells in two tothree rounds of division to form an entire sensory organon the notum (Fig. 4A). Wild-type flies have two scutel-lar bristles (Fig. 4B) on each heminotum, but 14.3% of

Figure 1. Generation of miR-9a loss-of-function mutant flies. (A) Nucleotide sequence alignment of miR-9a among different species.(B) The cytological location of miR-9a in the Drosophila genome. (C) The ends-out recombination approach was used to replace the78-nt long miR-9a precursor with the white gene. (D) The miR-9a mutant lines were identified by PCR analysis using primers flankingthe miR-9a locus. (E) Northern blotting analysis confirmed that miR-9a was absent in the mutant lines. The same Northern membranewas probed with a 2s rRNA probe as the loading control.

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miR-9a mutant flies (14 out of 98) had three or fourbristles (Fig. 4C) with accompanying shaft and socketcells (Fig. 4E). Conversely, flies overexpressing miR-9a in

the notum region by ap-Gal4 had a severe loss of scute-llar sensory organs (Fig. 4D). Like the wing margin phe-notype, defects on the notum of miR-9a mutant could be

Figure 2. Loss of miR-9a activity resulted in the formation of ectopic sensory neurons in Drosophila embryos and larvae. (A)Schematic representation of the dorsal cluster sensory neurons in each abdominal hemisegment. (B) Wild-type ddaE and ddaF neuronsin third instar larvae are labeled by mCD8-GFP with Gal4221. The white arrows in B, C, and D indicate axons of these sensory neurons.The red arrowheads indicate ddaC neurons that are faintly labeled by GFP with Gal4221. In miR-9a mutant larvae, duplicated ddaF (C)and ddaE (D) neurons (yellow arrowheads) were observed with near normal dendritic branches (traced in C�,D�). (E,F) All the neuronsof one dorsal cluster of stage 15 embryos were double-labeled with anti-ELAV (green) and anti-Cut (red) antibodies. (E) In wild-typeembryos, there are 12 ELAV+ cells, including MD neurons and ES neurons, and eight Cut+/ELAV− support cells. (F) A miR-9aJ22 embryowith 14 ELAV+ cells and 10 Cut+/ELAV− support cells. (G,H) miR-9a expression is detected in epithelial cells in wild-type embryoswith in situ hybridization. (G) After gastrulation at stage 7, miR-9a expression persists in most epithelial cells except the ventralectoderm. (H) At stage 12–13, miR-9a is expressed in the ectoderm, with higher levels at the segmental boundaries and stomodeum.

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rescued by expression of miR-9a precursors using vg-Gal4 (n = 152). To further demonstrate the bristle phe-notype was due to increased number of scutellar SOPs inmiR-9a mutants, we stained the notum with Elav anti-body. We found that each ectopic bristle was also asso-ciated with an extra neuron (Fig. 4E), supporting our hy-pothesis that miR-9a regulates the specification of SOPsduring sensory organ development.

miR-9a genetically interacts with sens

The wing margin phenotypes of miR-9a mutant flies areremarkably similar to that seen in Lyra1 mutant flies, again-of-function mutant of sens, and in flies with forcedexpression of Sens in the wing (Nolo et al. 2001). More-over, the sens 3�UTR contains miR-9a-binding sites(Stark et al. 2003), raising the possibility that Sens mightbe misregulated in miR-9a mutant flies. To test this hy-pothesis, we performed genetic interaction studies. In amiR-9aJ22 homozygous mutant background, removingone copy of sens (sensE58/+) led to a substantial rescue ofthe wing margin defect. About 30% of these flies hadwings indistinguishable from the wild type, while otherslost only a smaller part of the posterior margin (Fig. 3D);the bristles on the anterior margin also resembled wildtype (Fig. 3D�). In contrast, 100% of miR-9a mutant flieshad posterior wing margin defects (Fig. 3). These findingssuggest that up-regulation of Sens is a key downstreamevent caused by loss of miR-9a activity.

To provide further evidence that miR-9a regulatesSens during the sensory organ formation, we performedadditional genetic interaction experiments on the no-tum. Overexpressing Sens with the vg-Gal4 induced ec-topic formation of numerous bristles on the notum (Fig.4F). However, coexpressing of the miR-9a precursor andSens largely repressed ectopic bristle formation (Fig. 4G),indicating that miR-9a can suppress Sens expression invivo. In this experiment, the UAS–sens construct con-tains the sens 3�UTR. The bristle defect was rescued todifferent degrees with two different UAS–pre-miR-9atransgenic lines (lines #3 and #8) (Fig. 4H). As a control,overexpression of the mutant miR-9a precursor with a7-nt deletion failed to rescue the sens overexpressionphenotype (Fig. 4H). These data provide further evidencethat miR-9a and sens function in the same genetic path-way in controlling SOP formation.

miR-9a regulates Sens expression in vivo

To provide mechanistic insights into the actions of miR-9a in controlling sensory organ formation, we set out toexamine whether the Sens protein level could be affectedby miR-9a in vivo. In the Drosophila imaginal wing disc,sens is expressed in proneural clusters and accumulatesin presumptive SOPs (Nolo et al. 2000). We performed insitu hybridization on the wing discs of third instar larvaeand found that miR-9a is widely expressed except forsome cells in the wing margins in wild-type flies (Fig.

Figure 3. Wing margin defects in miR-9amutant flies. (A) A wild-type wing. Therectangle indicates the areas enlarged intoA� (same for those in B–D). (A�) Sensoryhairs on anterior wing margin are as-sembled in a regular pattern with fourmacrochaetes flanking one microchaete.(B) All miR-9a mutant flies lose the poste-rior wing margin, while the veins are nor-mal in appearance. (B�) Ectopic sensoryhairs on the anterior wing margin in miR-9a mutants. (C,C�) The wing margin de-fects in miR-9a were rescued by expres-sion of the wild-type miR-9a precursor(line #8) with vg-Gal4 (n = 152). (D,D�) Thewing margin defects of miR-9a mutantswere suppressed by removing one copy ofsens. sensE58 was recombined with miR-9aJ22 onto the same chromosome.





5A), but not miR-9a mutants (Fig. 5B). To examine miR-9a expression at the cellular level, we performed in situhybridization of miR-9a on a A101-Gal4/UAS–mCD8-GFP disc in which a subset of SOPs is labeled with GFP(Fig. 5C). Interestingly, miR-9a is not expressed in SOPs,but is expressed in most, if not all, epithelial cells, in-cluding the ones adjacent to SOPs (Fig. 5D,E). Sens isdynamically regulated within proneural clusters duringSOPs specification: It is up-regulated in SOPs and down-regulated in other cells (Nolo et al. 2000). The expressionpattern of miR-9a is consistent with the “avoidance hy-pothesis” that miR-9a may control SOP formationthrough regulation of gene expression in non-SOP cells(Stark et al. 2005).

To provide direct evidence that miR-9a plays a role inSOP formation by regulating Sens expression, we exam-ined Sens expression in the wing imaginal disc. Asshown by immunostaining of wing imaginal discs ofwild-type third instar larvae, Sens is first expressed inproneural cluster cells at an early stage and is concen-trated in SOPs during SOP specification. Two rows of

Sens+ cells at the pouch region develop into SOPs in thelate third instar stage and into sensory bristles on adultwing margins (Fig. 5F). In the region that develops intothe notum, there are two Sens+ cells, which are the pre-cursors for the two scutellar bristles on each hemi-no-tum (Fig. 5F, inset). In the miR-9a mutant, there weremore SOPs in early third instar wing discs (Fig. 5G), and>60% of the discs contained three or four Sens+ SOPs inthe notum region (Fig. 5G, inset). These findings are con-sistent with the ectopic bristles in adult miR-9a mutantflies (Fig. 4C).

Conversely, overexpression of miR-9a in the dorsalcompartment of the wing disc by ap-Gal4 (Calleja et al.1996) significantly reduced the number of Sens+ cells,and the two SOPs in the notum region were absent(Fig. 5H), consistent with the balding phenotype in adultmutant flies (Fig. 4D). These data provide strong evi-dence that the ectopic sensory organs in miR-9a mutantflies result from ectopic SOPs during neurogenesis, andthat miR-9a normally suppresses Sens expression, therebyinhibiting the SOP fate in surrounding epithelial cells.

Figure 4. miR-9a mutant flies show a sensory bristle defect on the notum, and miR-9a genetically interacts with sens. (A) Schematicrepresentation of the cell lineage of an adult sensory organ from a single SOP. (B) Wild-type flies have four scutellar bristles (asterisks)on the notum. (C) miR-9a mutant flies have four to seven scutellar bristles on the notum (asterisks). (D) Overexpression of miR-9aresulted in a loss of scutellar bristles (asterisk indicates the only remaining bristle). (E) In miR-9a mutant flies, all bristles (yellowarrowhead) have normal shaft and socket cells and are accompanied by ELAV+ sensory neurons (green arrowheads). Some bristles werelost during the staining process. (F) Overexpression of Sens dramatically increased the number of sensory bristles on the notum. (G)Coexpression of miR-9a suppressed the generation of ectopic bristles induced by Sens overexpression. (H) Statistical analysis of geneticinteractions between sens and miR-9a. Genotypes are as follows: wild-type (n = 50) (bar 1); vg-Gal4/+;UAS–sens/+ (n = 20) (bar 2);vg-Gal4/+;UAS–sens/UAS–pre-miR-9a (line #3) (n = 20) (bar 3); vg-Gal4/UAS–pre-miR-9a; (line #8) UAS–sens/+ (n = 20) (bar 4); andvg-Gal4/+;UAS–sens/UAS–mutant–pre-miR-9a (n = 20) (bar 5). Values are means ± SEM. (***) p < 0.001.

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Figure 5. miR-9a is expressed in epithelial cells, including cells adjacent to SOPs in wing imaginal discs. (A) miR-9a is ubiquitouslyexpressed in wild-type wing imaginal discs except the wing margins, as shown with a Dig-labeled probe detected with an AP-conjugated anti-Dig antibody. (B) A miR-9aJ22 mutant wing imaginal disc has no miR-9a expression. (C–E) High-resolution fluorescentin situ hybridization shows that miR-9a is expressed in the epithelial cells, including cells adjacent to the SOPs in the pouch regionof a wild-type wing imaginal disc. (C) At the wing margins, SOPs were visualized by mCD8-GFP driven by A101-Gal4 (yellowarrowheads indicate two of them). (D) A Dig-labeled miR-9a probe detected by Cy3-conjugated anti-Dig antibody (red) shows miR-9ais expressed in the epithelial cells, including ones adjacent to the SOPs. (E) Merged image of C and D showing miR-9a is excluded fromthe SOPs in the wing margins. (F–H) Immunostaining of Sens (red) in wing imaginal discs of early third instar larvae. (F) In thewild-type wing disc, Sens is highly expressed in SOPs and wing margins. Inset shows a magnified view of the notum region from whichscutellar bristles are derived. (G) miR-9aJ22 mutant imaginal wing disc has ectopic SOPs. (Inset) A high frequency of extra SOPs wasobserved in the notum region. (H) When miR-9a is overexpressed with ap-Gal4 in the dorsal part of the wing disc, Sens expression wassuppressed in most of the dorsal compartment. Inset shows the absence of Sens+ cells (SOPs) in the region that develops into thenotum.





miR-9a directly suppresses sens expressionthrough its 3�UTR

It was predicted that the sens 3�UTR contains three miR-9a-binding sites (Fig. 6A; Stark et al. 2003). To testwhether miR-9a suppresses Sens expression directlythrough its 3�UTR, we performed luciferase assays.HEK293 cells were cotransfected with the wild-typemiR-9a precursor and sens 3�UTR downstream from theluciferase coding region. The relative luciferase activitywas 70% lower than in cells expressing the luciferase-sens 3�UTR alone (Fig. 6C). Two mutant constructs ofmiR-9a precursors were generated as controls, one witha 5-nt deletion in the mature miR-9a region (Mut-1) andone with a 7-nt deletion in the stem of the precursor(Mut-2) (Fig. 6B). Both of these mutant constructs failedto suppress the expression of luciferase with the sens3�UTR (Fig. 6C). As an additional control, none of themiR-9a constructs had any effect on the expression ofluciferase without the sens 3�UTR (data not shown).These findings provide strong evidence that miR-9adirectly suppresses sens expression through its 3�UTR.

Discussion

Drosophila miR-9a suppresses neuronalprecursor specification

miR-9a is one of the miRNAs that are highly expressedin the mammalian brain and 100% conserved at the

nucleotide sequence from flies to humans, suggesting animportant role in brain development and/or function (La-gos-Quintana et al. 2002; Aravin et al. 2003; Sempere etal. 2004). We generated miR-9a loss-of-function allelesand found that homozygous mutant flies developed intoadulthood at the expected Mendelian ratio. Adult mu-tant flies are grossly normal and fertile, indicating thatmiR-9a is not required for viability or fertility. This find-ing is different from the reported severe dorsal closuredefects and embryonic lethal phenotype generated by an-tisense 2�O-methyl oligoribonucleotide-mediated deple-tion of miR-9a (Leaman et al. 2005).

Interestingly, Drosophila miR-9a is not expressed inmature neurons, but is expressed in epithelial cells, in-cluding the proneural clusters that give rise to SOPs(Figs. 2, 5; Stark et al. 2005). Detailed analysis of embry-onic PNS development revealed an unexpected findingthat miR-9a mutants have an increased number of sen-sory neurons (Fig. 2) that elaborate extensive dendriticarbors underneath the epithelial cell layer, such as ddaEand ddaF neurons (Sweeney et al. 2002). The duplicatedneurons occupy the same dendritic field and appear tohave similar dendritic branching patterns (Fig. 2C,D). In-deed, the average numbers of dendritic ends of ddaE andddaF neurons in abdominal segments 3–5 were similar inwild-type and miR-9a mutant larvae and MARCMclones (data not shown), indicating that loss of miR-9aactivity affected the number of these sensory neuronsonly but had no cell-autonomous effect on their den-dritic branching patterns.

The effect of miR-9a on the number of embryonic sen-

Figure 6. Luciferase assay showing the suppression by miR-9a through the sens 3�UTR. (A) The sequences and locations of threepredicted miR-9a-binding sites in the sens 3�UTR and their alignment with miR-9a (red). (B) The predicted stem-loop-stem structureof the miR-9a precursor. (WT) Wild-type precursor; (Mut-1) mutant precursor 1 with a 5-nt deletion in mature miR-9a; (Mut-2) themutant precursor with a 7-nt deletion in the stem region complementary to mature miR-9a. Mature miR-9a is highlighted in red, andstrikethroughs indicate the deleted nucleotides. (C) Wild-type (WT) but not mutant miR-9a precursors (Mut-1 and Mut-2) couldsuppress the expression of luciferase with the sens 3�UTR. Bars indicate the average of luciferase activity in one triplicated experiment.(***) p < 0.001.

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sory neurons has two major features. First, the ectopicddaE or ddaF neurons were generated as a result of ecto-pic SOPs and not cell fate transformation within a celllineage (Fig. 2), suggesting miR-9a affects an early step inneurogenesis, consistent with its embryonic expressionpattern (Fig. 2; Stark et al. 2005). Second, both the ex-pressivity and penetrance of this defect were relativelylow. This finding supports the idea that miRNAs, atleast in this particular case, are not developmentalswitches, but instead function as a fine-tuning mecha-nism to ensure the accuracy of a particular developmen-tal process (e.g., Farh et al. 2005; Stark et al. 2005).

In this study, we focused on the formation of SOPs inadult flies. Like embryos, only ∼14% of miR-9a mutantflies exhibited ectopic SOPs on the notum, again indi-cating a fine-tuning role for miR-9a in controlling SOPformation. However, our analysis of the miR-9a mutantphenotype in adult flies also indicates that miRNAs canhave dramatic effects on some other developmental pro-cesses. For instance, miR-9a is widely expressed in thewing disc (Fig. 5), and 100% of miR-9a mutant flies ex-hibited a severe posterior wing margin defect (Fig. 3),suggesting that cell proliferation and/or survival aremuch more sensitive to changes in the expression levelsof the proteins regulated by miR-9a.

MiR-9a inhibits Sens expression in non-SOP cells

How does miR-9a exert its effect on SOP formation?Sens is a zinc finger transcription factor required tomaintain high-level expression of proneural gene inSOPs and to suppress their expression in non-SOP cells(Nolo et al. 2000; Jafar-Nejad et al. 2003). Several find-ings in this study demonstrate that Sens is a key target ofmiR-9a regulation and is essential for mediating miR-9afunction in SOP formation. (1) The wing margin defectsin miR-9a mutant flies were remarkably similar to thatcaused by overexpression of Sens by the UAS-Gal4 sys-tem or in Lyra1 mutants (Nolo et al. 2000, 2001). (2)miR-9a was expressed at a much lower level in SOPsthan in adjacent epithelial cells, correlating with thehigh level of Sens expression in SOPs and the low level ofSens in non-SOP cells in proneural clusters. The inabil-ity to use immunostaining to detect subtle changes ofSens expression level in non-SOP cells due to miR-9aloss of function could be attributed to the following rea-sons: Sens expression is primarily down-regulated at thetranscriptional level in the non-SOP proneural cells, andmiR-9a’s function is limited to preventing translation ofthe leaky/residual sens mRNA, consistent with themodel proposed by Stark et al. (2005). The alteration in

Figure 7. A model for the role of miR-9a inregulating SOP specification. Within one proneu-ral cluster, the cell selected to become the SOPexpresses a high level of Delta to activate Notchin the adjacent cells (non-SOPs). Upon activa-tion, Notch releases the intracellular domain(NIC), which translocates to nuclei and binds toSuppressor of Hairless, switching it to the activeform [Su(H)Act]. The NIC/Su(H)Act complex acti-vates Enhancer of Split [E(spl)], and consequentlyrepresses sens transcription. In SOPs, withoutthe activation of Notch, Su(H) functions as a re-pressor [Su(H)Rep] to suppress the E(spl). There-fore, sens could be continuously transcribed, al-lowing high-level accumulation of Sens protein,which is required for further transcription of neu-ron-specific genes. miR-9a functions in non-SOPcells to further suppress Sens translation throughits 3�UTR to ensure the precise production ofSOPs.





Sens level, due to loss of miR-9a function in the non-SOP cells, is sufficient to initiate the production of ec-topic SOPs, but it may not be dramatic enough to bedetected by immunostaining. (3) The sens 3�UTR con-tains three miR-9a-binding sites and is the best predictedtarget of miR-9a (Stark et al. 2003). (4) Wild-type but notmutant miR-9a precursors down-regulated reporter geneexpression through the sens 3�UTR in transfected cells.(5) Overexpression of miR-9a in vivo inhibited Sens ex-pression. We observed that Sens expression along thewing margin in the dorsal compartment was lower thanin the ventral compartment in some wing discs whenmiR-9a was expressed by ap-Gal4 (data not shown). Thefailure to completely suppress Sens expression in thedorsal compartment (Fig. 5H) is probably due to the factthat, at this developmental stage, Sens expression in thewing margins is controlled by proneural genes, unlikeSOPs in the notum region where Sens expression ismaintained by itself (Jafar-Nejad et al. 2003). (6) miR-9aand sens showed strong genetic interactions in control-ling SOP formation.

These findings provide an experimental example tosupport the notion that miRNAs and their mRNA tar-gets are often expressed in cells adjacent to each other(Stark et al. 2005). The differential expression of Sens inSOPs and adjacent neuroepithelial cells is essential forthe production of a precise number of SOPs during de-velopment (Nolo et al. 2000; Jafar-Nejad et al. 2003). Wepropose a model in which miR-9a functions in non-SOPscells to further suppress Sens expression at the transla-tional level, as a complementary mechanism to the tran-scriptional inhibition of Sens expression by E(spl) (Fig. 7).Loss of miR-9a function increases Sens protein level, notso dramatically but just enough to convert Sens in someneuroepithelial cells from a transcription repressor intoan activator of proneural genes, therefore resulting in theformation of a small number of ectopic SOPs. However,unlike many other genes essential for neurogenesis, suchas Notch and Delta, miR-9a does not function as an ab-solute switch. Instead, it only ensures the accurate dif-ferential Sens expression and fine-tunes this develop-mental process.

Overexpression of miR-9a in the wing imaginal disccould dramatically inhibit the formation of sensory or-gans on the notum (Fig. 4D), suggesting that misregula-tion of miR-9a expression itself could potentially havesevere developmental consequences. Since both miR-9aand E(spl) have similar functions in non-SOP cells, it ispossible that both genes may be regulated by a similartranscriptional mechanism. Indeed, we noticed thatbinding sites for the Achaete–Scute complex and Su(H)are present in the regulatory region of miR-9a. Takentogether, our studies presented here have uncovered an-other layer of gene regulation during early neurogenesisin the Drosophila PNS. miR-9a is 100% conserved at thenucleotide level from flies to humans. Moreover, the hu-man miR-9a is highly expressed in fetal but not in adultbrains (Nelson et al. 2006). Therefore, a similar mecha-nism of miR-9a function may operate during mamma-lian neurogenesis as well.

Materials and methods

Fly strains and genetics

All the flies were maintained at 22°C–25°C on standard me-dium. The w1118 strain was used as a wild-type control. Gal4221

combined with UAS–mCD8�GFP was used to visualize indi-vidual ddaE and ddaF neurons in the larval PNS. It was recom-bined with miR-9a mutants to generate the homozygous stockGal4221, UAS–mCD8�GFP, miR-9a. For studies of the wingand the notum, the following lines were used: vg-Gal4, ap-Gal4(Calleja et al. 1996), and UAS–sens (Nolo et al. 2000). The pres-ence of sens 3�UTR in the UAS–sens construct was confirmedby PCR. sensE58 was used as the sens loss-of-function mutantallele in genetic interaction experiments.

Target homologous recombination constructs

miR-9a loss-of-function mutations were generated with theends-out technology (Gong and Golic 2003). The targeting con-struct contains the w+ gene flanked by two arms of genomicDNA fragments at the miR-9a locus. The two arms were pre-pared by PCR from the genomic DNA using primers 5�-AGC-GATCGTCGTCGGACTAC-3� and 5�-ACACTGCAGATGGT-TGAAAG-3� for the left arm, and primers 5�-ACTCGAGC-CAAAAACGAGGCCCACA-3� and 5�-AGGTACCGAGACAGCAAAATCGTAGAA-3� for the right arm. Two independenttransgenic fly lines carrying the targeting construct werecrossed to hs-FLP, I-SceI (Bloomington Stock Center), and theprogenies at late second to third instar stages were heat-shockedfor 45 min at 38°C. Virgin female flies with mosaic eyes werecollected and crossed to p{70FLP}10 (Bloomington Stock Cen-ter), and at the next generation, individual males with solid-color eyes were crossed to TM3 Ser/TM6 Tb as candidate lines.For the replacement of the miR-9a by w+, a PCR screen wasperformed with genomic DNAs isolated from each line. North-ern blot was used to confirm the absence of miR-9a expressionin mutant flies.

Transgenic constructs

The 78-base-pair (bp) miR-9a precursor was cloned from wild-type genomic DNA with primers 5�-GAATTCTATACAGGGT-GCTATGTTG-3� and 5�-TCTAGACGCTGGGCAGACGC-TAATATTAACTTCGG-3�. The mutant precursor with a 7-bpdeletion on the stem was generated with primers 5�-GAATTC-TATACAGGGTGCTATGTTG-3� and 5�-TCTAGACGCTGGCAGACGCTAATATTAAAAGCTAGCTTTATGACGT-3�.Both fragments were subcloned into the pUAST vector and in-jected into embryos to generate transgenic fly stocks that ex-press either wild-type or mutant miR-9a precursor under thecontrol of UAS elements.

Northern blot

Total RNA was extracted from larvae with Trizol, according tothe manufacturer’s instructions (Invitrogen). For each sample,20 µg of total RNA was run on a 12% polyacrylamide gel (Se-quagel, National Diagnostics) and transferred to a Nytran Su-perCharge Signal membrane (Schleicher & Schuell). miR-9awas detected with a purified 32P-end-labeled oligo probe, 5�-TCATACAGCTAGATAACCAAAGA-3�, which is comple-mentary to the mature miR-9a sequence.

In situ hybridization

Embryos and wing imaginal discs were fixed and stained accord-ing to standard procedures (Tautz and Pfeifle 1989). A genomic

Li et al.




fragment of 1 kb flanking the miR-9a precursor was obtained byPCR and cloned into the pBSK vector. A Digoxigenin (Dig)-labeled riboprobe was generated by in vitro transcription of thisconstruct with T7 RNA polymerase. A Drosophila miR-9alocked nucleic acid (LNA) probe was obtained from Exiqon(Vedbaek), end-labeled with a Dig oligonucleotide 3�-end-label-ing kit (Roche), and purified on a G-25 Microspin column (Am-ersham Biosciences). In situ hybridization with both probes wasdetected by AP-conjugated cy3-conjugated anti-Dig antibody ac-cording to standard procedures (Tautz and Pfeifle 1989) with thefollowing modifications: After post-fixation and washes, thediscs were washed with water and treated twice for 5 min eachwith 0.25% (v/v) acetic anhydride in 0.1 M triethanolamine (pH8.0). The tissues were prehybridized and hybridized at 50°C inhybridization buffer (pH 6.0, adjusted with citric acid).

Antibody staining

Embryos were fixed according to the standard protocol. Thirdinstar larval wing imaginal discs were fixed in 4% formaldehydein PBT (PBS and 0.1% Triton X-100) for 30 min at room tem-perature. Pupae were dissected 48 h after pupa formation andfixed in 4% formaldehyde in PBT overnight at 4°C. The follow-ing pairs of primary and secondary antibodies were used for allexperiments: anti-Sens (1:1000) (Nolo et al. 2000) and anti-guinea pig-Cy3 (1:500; Molecular Probes); anti-ELAV (1:100; De-velopmental Hybridoma Study Bank) and anti-mouse-Cy3 oranti-mouse-FITC (1:300; Jackson Laboratory); anti-Cut (1:1000;Developmental Hybridoma Study Bank) and anti-rabbit-Cy3(1:300; Jackson Laboratory).

Microscopy and imaging

All samples with fluorescent signals were imaged by confocalmicroscopy (Nikon, D-Eclipse C1), including GFP-labeled DAneurons in third instar larvae; immunostaining on embryos,wing discs, and the pupa notum; and fluorescence-labeled insitu hybridization on wing discs. Images of adult wing and no-tum and Dig-labeled in situ hybridization on wing discs wereobtained with an Olympus BX60 microscope and AxioCam Hrccamera.

Luciferase reporter assays

The wild-type and two mutant miR-9a precursors were ob-tained by PCR and cloned into the pSUPER vector under thecontrol of the H1 promoter (OligoEngine). A 600-bp fragment ofthe sens 3�UTR was cloned into the pGL3-promoter vector (Pro-mega) downstream from the firefly luciferase gene. The Renillaluciferase plasmid served as the transfection control. HEK293cells were cotransfected in 12-well plates with three vectors (0.5µg of each vector per well): the luciferase-sens 3�UTR reporterplasmid, the Renilla luciferase plasmid, and the pSUPER vectorcontaining miR-9a precursor (wild type, Mut-1, or Mut-2) or thevector only. Three independent transfections with triplicateseach time were performed, and cell lysates were collected 28 hafter transfection. Relative luciferase activity was measuredwith Dual-luciferase assays (Promega) according to the manu-facturer’s protocol.

Acknowledgments

We thank H. Bellen, the Bloomington Stock Center, and theDevelopmental Hybridoma Bank for reagents and fly lines. Wethank S. Ordway and G. Howard for editorial assistance, K.

Nyuyen for manuscript preparation, and L.-P. Chang for helpwith the UAS–pre-miR-9a construct. We also thank T. Korn-berg, Y. Hong, and laboratory members for discussions and com-ments during the course of this work. This study was supportedby an NIH training grant (T32 AG00278 to F.W.), the KoreaResearch Foundation (KRF: M01-2005-000-10157-0 to J.L.), andthe Esther A. and Joseph Klingenstein Fund, the McKnight En-dowment Fund for Neuroscience, FRAXA Foundation, Pfizer/AFAR, and the NIH (to F.-B.G.).

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